RSV is the most frequent cause of the hospitalization of unweaned infants under one year old for acute respiratory infections. Infants suffering from laryngotracheobronchites, bronchiolites and pneumonias require hospital care, and the incidence of mortality in unweaned infants exhibiting congenital cardiac diseases is greater than 37%. Other disorders such as bronchopulmonary dysplasias, renal diseases and immunodeficiency are equally much factors which are responsible for increased mortality. Infections with RSV can also be a cause of death in old people.
In temperate countries, the RSV epidemic occurs during the winter period from November to April, and the highest incidence of serious diseases is found in the unweaned infant of 2 to 6 months. A distinction is made between two types of RSV, RSV-A and RSV-B, on the basis of the antigenic variation of the G glycoprotein of RSV: subgroup A and subgroup B, which circulate concurrently. A recent study which was carried out in France from 1982 to 1990 demonstrated the alternation of one subgroup with the other over a period of 5 years. Strain A is often the cause of infections which are more serious than those caused by strain B.
In the 1960""s, an unsuccessful attempt was made to develop conventional vaccines, that is using formolinactivated RSV, in analogy with anti-measles vaccines. Instead of conferring protection on the vaccinated infant, this type of vaccine had the effect of potentiating the natural viral disease.
Human RSV belongs to the genus pneumovirus, which is a member of the Paramyxoviridae family. The genome of the virus consists of an RNA strand which is of negative polarity, is nonsegmented and encodes 10 distinct proteins: NS1, NS2, N, P, M, SH (or 1A), G, F, M2 (or 22K) and L.
Numerous published experiments have demonstrated that the main proteins involved in protection are: F, G and N. The fusion glycoprotein F, which is synthesized as precursor F0, is cleaved into two subunits F1 (48 kDa) and F2 (20 kDa) which are bound together by disulphide bridges. The F protein is conserved between RSV-A and RSV-B (91% homology). Conversely, the attachment glycoprotein G varies greatly from one subgroup to the other. Only one region of 13 amino acids (aa 164 to aa 176) is highly conserved and four cysteine residues (173, 176, 182 and 186) are preserved in each subgroup. It has been shown in animal models that the two glycoproteins F and G play a major role in the immunology of RSV. Monoclonal antibodies directed against G and F are able to neutralize the virus in vitro and, when administered passively, they protect the cotton rat from RSV infection.
Current treatments for aggravation of the disease caused by RSV in unweaned infants are clearing the respiratory tract of congestion by aspirating mucus and respiratory assistance provided by ventilation. An antiviral agent, ribavirin, appears to be effective in seriously affected cases. However, its use in paediatric therapy is still poorly defined. Passive immunization with anti-RSV immunoglobulins represents an alternative route in the treatment of serious RSV infections: no undesirable side-effect has been observed. Nevertheless, this type of treatment is very costly and difficult to extrapolate to a large scale.
Different approaches have been taken to vaccinating against human RSV: either the vaccine protects against RSV infection in animals (rodents and primates) but induces pulmonary pathology or else the vaccine is not sufficiently immunogenic and does not provide protection (Connors et al., Vaccine 1992; 10: 475-484).
For this reason, the present invention relates to a process for improving the immunogenicity of an immunogen, in particular an antigen, or a hapten, when it is administered to a host, independently of the mode of administration, characterized in that the said immunogen or hapten is coupled covalently to a support molecule in order to form a complex, and in that this support molecule is a polypeptide fragment which is able to bind specifically to Tmaimalian serum albumin.
Administration can, in particular, take place enterally, parenterally or orally.
The immunogenicity of the complex between the immunogen and the support molecule is found to be improved as compared with that of the immunogen alone, in the absence of any other immunostimulant.
A complex which is particularly suitable for implementing the present invention is obtained by using a conjugate with a polypeptide which is derived from the G protein of Streptococcus; this protein has been characterized by Nygren et al. (J. Mol. Recognit. 1988; 1:69-74).
The invention relates to a process in which the support molecule exhibits the amino acid sequence denoted sequence ID No: 74 or a sequence which exhibits at least 80%, and preferably at least 90%, homology with the said sequence ID No: 74.
This sequence can be attached to linking sequences which promote its expression in a host.
According to the invention, use can also be made of a support molecule which exhibits one of the sequences ID No: 75 or No: 78, as well as of molecules which exhibit at least 80%, and preferably at least 90%, homology with the said sequences.
The peptide sequence ID No: 78 exhibits the following characteristics:
The complex between the support molecule and the compound whose immunogenicity it is desired to improve can be produced by recombinant DNA techniques, in particular by inserting or fusing the DNA encoding the immunogen or hapten into the DNA molecule encoding the support.
According to another embodiment, the covalent coupling between the support molecule and the immunogen is effected chemically using techniques known to the person skilled in the art.
The invention also relates to a gene fusion which renders it possible to implement the process for improving the immunogenicity, characterized in that it comprises a hybrid DNA molecule which is produced by inserting or fusing the DNA encoding the immunogen or hapten into the DNA molecule encoding the support molecule and which is fused with a promoter; the invention also comprises a vector which contains such a gene, it being possible for the said vector to have, in particular, as its origin a DNA vector which derives from a plasmid, a bacterio-phage, a virus and/or a cosmid.
A vector which exhibits the sequence ID No: 76 or 77 belongs to the invention, as does the corresponding polypeptide. These polypeptides exhibit the following characteristics:
The DNA molecule which encodes the complex between the immunogen and the support molecule can be integrated into the genome of the host cell.
In one of its embodiments, the novel process includes a step for producing the complex, by genetic manipulation, in a host cell.
The host cell can be of the prokaryote type and be selected, in particular, from the group comprising: E. coli, Bacillus, Lactobacillus, Staphylococcus and Streptococcus; it can also be a yeast.
According to another aspect, the host cell is derived from a mammal.
The gene fusion which encodes the complex having an improved izmunogenicity can, in particular, be introduced into the host cell by the mediation of a viral vector.
The immunogen which is used preferably derives from bacteria, parasites and viruses.
This immunogen can be a hapten: peptide or polysaccharide.
The novel process is particularly suitable for a surface polypeptide from a pathogen. When this polypeptide is expressed in the form of a fusion protein, using recombinant DNA techniques, the fusion protein is advantageously expressed, anchored and exposed at the surface of the membrane of the host cells. Nucleic acid molecules are employed which are able to direct the synthesis of the antigen in the host cell.
They comprise a promoter sequence, a functionally linked secretion signal sequence and a sequence which encodes a membrane anchoring region, which sequences will be adapted by the person skilled in the art.
The immunogen can, in particular, be derived from an RSV surface glycoprotein: F and/or G.
Particularly advantageous results are obtained using fragments of the G protein from RSV subgroups A or B.
The proteins which are derived from the G glycoprotein of RSV subgroup A and subgroup B can be genetically fused or chemically coupled to BB.
The invention relates, therefore, to a complex which is obtained using the sequence which is encompassed between amino acids 130 and 230 of the G protein of RSV, or a sequence exhibiting at least 80% homology with the said sequence of the G protein.
This sequence can be obtained from human or bovine RSV which belongs to subgroups A or B.
The sequence encompassed between amino acids 130 and 230 of the G protein can be subjected to various types of modification for the purpose of modulating its immunogenic activity and its expression by the host system.
The applicant has, in particular, demonstrated that polypeptides are of interest in which:
the Cys amino acid in positions 173 and/or 186 has been replaced by an amino acid which does not form a disulphide bridge, in particular serine, and/or
the amino acids in positions 176 and 182 are capable of forming a covalent bridge other than a disulphide bridge, in particular aspartic acid and ornithine, and/or
the phenylalanine amino acids corresponding to positions 163, 165, 168 and/or 170 of the sequence of the G protein are replaced by a polar amino acid, in particular serine, and/or
the sequence encompassed between the amino acids numbered 162 and 170 is deleted.
Peptides exhibiting one of the sequences ID No: 1 to 73, or a sequence possessing at least 90% homology with one of the sequences ID No: 1 to 73, are thus particularly suitable for implementing the invention.
Other immunogens which are suitable for implementing the novel process include a derivative of the surface protein of hepatitis virus A, B and C, a surface protein of the measles virus, a surface protein of parainfluenza virus 3, in particular a surface glycoprotein such as haemagglutinin, neuraminidase HN and fusion protein F.
The RNA or DNA nucleotide sequences which encode complexes such as those previously defined, and which include elements which enable expression to be targeted in certain specific host cells, are included in the invention. They can be incorporated into a viral or plasmid vector; this vector will be administered to a mammal, in particular within a pharmaceutical composition, in order to enable the complex between the immunogen and the support molecule to be produced in situ.
The invention also relates to the use of a gene fusion or a complex between an immunogen (P) and a support molecule, such as those previously defined, as a medicament. The pharmaceutical compositions containing the gene or the complex together with physiologically acceptable excipients also belong to the invention. They are particularly suitable for preparing a vaccine.
Immunization can be obtained by administering the nucleotide sequence either on its own or through the agency of a viral vector. The host cell, in particular an inactivated bacterium, may also be used. Finally, the complex which is obtained by chemical coupling or is in the form of a fusion protein induces an antibody response which is very powerful compared with that induced by (P) on its own coupled to Freund""s adjuvant.
Within the scope of a vaccine against RSV, the applicant has demonstrated the efficacy of the fusion protein BBG2A, where G2A is a 101 amino acid fragment of the G protein of RSV-A (G aa 130-aa 230), seq id No. 1. When used to immunize rodents, BBG2A and BBG2Axcex4C coupled to alum (aluminium hydroxide) confer complete protection against challenge with RSV-A (Long strain).
The examples which follow are intended to illustrate the invention without limiting its scope in any way.